(Supported in part by NSF MCB 9420772 to B. McEwen). Chromosomes attach to the mitotic spindle via their kinetochores. The average number of spindle microtubules binding to each kinetochore varies with species, the stage of mitosis, and the length of time that the kinetochore has been attached to the spindle. In this study we used 3D EM and STERECON to investigate how kinetochore microtubule number varies with kinetochore size and chromosome size in PtK1 cells. From an analysis of serial section electron micrographs, we determined that the average surface area of metaphase, taxol-treated metaphase, and anaphase kinetochores is 0.16 +/- 0.05 (m2 (N = 181). Surprisingly, kinetochore microtubules are packed more densely on the smaller kinetochores, as seen by a reduction in the average spacing between kinetochore microtubules from 89 to 59 nm. Our interpretation of this result is that PtK1 cells require a minimum kinetochore microtubule-binding capacity for surv ival durin g repeated rounds of mitotic division. We estimate the lower limit to be 23 kinetochore microtubules and suggest that this capacity is required to ensure stable attachment during the dynamic and highly stochastic process of kinetochore fiber formation. There is a modest but statistically significant increase in kinetochore microtubule number with chromosome size, indicating that chromosome size is a minor determinant of kinetochore microtubule number. McEwen, B.F., Y. Ding and A. B. Heagle. (1998) Relevance of kinetochore size and microtubule-binding capacity for stable chromosome attachment during mitosis in PtK1 cells. Chromosome Research 6:123-132.